Role of the Highly Conserved LWYIK
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Role of the Highly Conserved LWYIK Motif of the HIV-1 Transmembrane Protein gp41 in Env-mediated Membrane Fusion. Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan R.O.C. August 14, 2006 2006 International AIDS Conference. FP. NHR. CHR.

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Steve S.-L. Chen and Woan-Eng Chan Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan

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Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Role of the Highly Conserved LWYIK Motif of the HIV-1 Transmembrane Protein gp41 in Env-mediated Membrane Fusion

Steve S.-L. Chen and Woan-Eng Chan

Institute of Biomedical Sciences

Academia Sinica

Taipei, Taiwan

R.O.C.

August 14, 2006

2006 International AIDS Conference


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

FP

NHR

CHR

TM

The Tryptophan-rich domain

Ectodomain

Cytoplasmic

domain

705

684

587

628

663

527

559

512

N

C

C34

DP178

(638-673)

663

705

679

683

ELDKWASWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIV

2F5

4E10

Membrane-spanning domain

(684-705)


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

The LWYIK motif (residues 679~683):

located immediately proximal to the TM region

Li and Papadopoulos: Cholesterol-binding motif: L/V-(X)1-5-Y-(X)1-5-R/K

HIV-1:

92BR025-9WQNLWTWFGITNWLWYIK

GB8.C4WANLWNWFDITNWLWYIK

NL4-3WASLWNWFNITNWLWYIK

MNWASLWNWFDITNWLWYIK

HXB2WASLWNWFNITNWLWYIK

SIV:

SIV cpzantWSSLWNWFDITQWLWYIK

SIV cpzLNSWDVFGNWFDLASWIR

SIV macLNSWDVFGNWFDLTSWIK

SIV agmLNSWDVFGNWFDLASWIK

HIV-2

HIV2CBL24LNSWDVFGNWFDLASWIK

HIV-2STLNSWDVFGNWFDLTSWIK

HIV2CBL21LNSWDVFGNWFDLTSWIR


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Vincent et al.Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups. Biochim. Biophys. Acta 1567: 157-164, 2002

  • The LWYIK motif in the format of a MBP fusion protein was shown to bind to cholesteryl group in vitro.

  • This motif was therefore proposed to play a role in Env association with lipid rafts and Env-mediated membrane fusion.

  • However, the biological significance of this motif in virus replication is not known.


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Construction of LWYIK motif-mutant proviruses

679 683

TM region

WT

----ITNWLWYIKLFIMI----

--------.....---------

DLWYIK

----------..----------

DYI

-----------..---------

DIK

KE

------------E---------

WA

---------A------------

YA

----------A-----------


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Replication kinetics of LWYIK

motif-mutant viruses

in CD4+ CEM-SS cells


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

All mutant viruses inhibit their

one-cycle virus infectivities: on an

HIV-1 LTR-driven,

cat gene-harboring reporter cell line, H938


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Mutations in the LWYIK motif do not have

significant effects on Env precursor synthesis,

processing, or Env incorporation into the virus


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Mutations in the LWYIK motif do not affect cell surface expression or CD4-binding ability of the Env


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

All of the mutant proteins are able to self-assemble into an oligomeric structure


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Env trans-complementation assay

rev

gag

CAT

vif

tat

SV40

pHXB

D

env

CAT

pol

D

env

LTR

LTR

Bgl

Bgl

rev

env

LTR

LTR

SV40

env

plasmid

gp120

gp41

45

753

314

363

517

Bgl

Bgl

Transfection

293

T cells

2days

Infection

+

CD4

T cells

CAT assay


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

All of mutant proteins inhibit

their trans-complementation abilities


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

A quantitative Env-mediated membrane fusion assay

CMV promoter

Tat

pA

Tat

HIV-1 5’-LTR

Cat gene

3’-LTR

Tat

293T

H938

Env

CD4


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Effects of mutations in the LWYIK motif

on the Env membrane fusion ability


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Mutations in the LWYIK motif have no

apparent effects on Env association with

lipid rafts


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Three-color dye transfer assay

CEM-SS

Env-expressing

293T cells

Dil andCalcein-AMlabeled

CMAClabeled

Hemifusion

Fusion pore enlargement


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Lipid and cytosolic mixing


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Deletions in the LWYIK motif

inhibit cytosolic dye transfer


Comparison of env mutants

Comparison of Env mutants

_________________________________________________________________

Env Membrane Virus

Incorporation fusion Infectivity Dye transfer

_________________________________________________________________________

WT Normal 100% 100% +

665~682 Greatly Reduced AbrogatedGreatly reduced -

W(1~5)A Greatly Reduced AbrogatedGreatly reduced +

W(1~3)A Greatly Reduced AbrogatedGreatly reduced

678~682 Greatly Reduced 20%Greatly reduced +

666~670 Greatly Reduced 20%Greatly reduced

LWYIK Normal - 10%No cytosolic mixing no cytosolic mixing

YI Normal - 10%Reduced cytosolic

mixing

IK Normal - 10%Reduced cytosolic

mixing

___________________________________________________________________________


Conclusion

Conclusion

  • While the LWYIK motif is not critical for Env localization in lipid rafts, this motif is critical for membrane fusion.

  • Env localization in lipid rafts does not necessarily warrant the membrane fusion ability of Env.

  • The LWYIK motif acts as a unique and distinct membrane fusion determinant located in the gp41 C-terminal ectodomain in modulating the membrane fusion process.


Acknowledgements

Acknowledgements

Institute of Biomedical Sciences, Academia Sinica:

Woan-Eng Chan

Hsiao-Fen Li

Yu Tsai

Shu-Chen Huang

Chia-Hung Chang

Animal Technology Institute Taiwan:

Chin-Kai Chuang

Supported by :

Academia Sinica and

National Health Research Institute

Taiwan, R.O.C.


The pre transmembrane region

The pre-transmembrane region

  • The pre-transmembrane region has been proposed to serve as

    • a flexible extender;

    • a contributor to trimer formation and stability; and

    • an agent for membrane destabilization that fosters membrane fusion.

  • Although Eric Hunter’s group previously implicated this Trp-rich region in membrane fusion and viral infectivity, the molecular basis for the role of this Trp-rich region in viral replication is still not fully understood.


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

A. Saez-Cirion et al. Biophysical J. 85:3769-3780, 2-003

Only the functional pre-TM sequence:

1), adopts helical structures in solution and in membranes;

2), forms homo-oligomers in solution and membranes; and

3), inhibits gp41-induced cell-cell fusion.

These data support two roles for gp41 aromatic-rich pretransmembrane sequence:

1), oligomerization of gp41; and 2), immersion into the viral membrane interface.

T. Suarez et al. FEBS Lett. 477:145-149, 2000

A functional peptide can induce vesicle leakage and lipid mixing.

A sequence representing a defective gp41 phenotype unable to mediate both cell–cell

fusion and virus entry, is equally unable to induce vesicle fusion, and adopted a

non-helical conformation in the membrane.

Therefore, membrane perturbation and adoption of the α-helical conformation by this

gp41 region might be functionally meaningful.

Salzwedel et al. J. Virol. 73:2469-2480, 1999

The multiple effects of various mutations in this region on membrane

fusion, Env incorporation into the virus, and virus infectivity suggest that different

residues or sequences in this motif may still play disparate functions in HIV-1

infection and that multiple sequences in this motif may contribute synergistically to

these functions.


Steve s l chen and woan eng chan institute of biomedical sciences academia sinica taipei taiwan

Membrane fusion- a series of cascade events:

Fusion of outer leafets

Fusion of inner leaflets

Fusion pore formation and enlargement


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